The high mobility group protein HMGB1 is an important mediator of the body's inflammatory, ischemia and injury. The HMGB1 protein contains a consensus the retinoblastoma (Rb) protein-binding motif LXCXE. RB is a well-characterized tumor suppressor, which plays an important role in regulation of proliferation, apoptosis and cell cycle progression in human breast cancer. Our preliminary studies have demonstrated that the HMGB1 protein interacts with RB via an LXCXE-dependent manner and increases the ability of RB in transcription "expression. More importantly, an increase of HMGB1 expression inhibits breast cancer cell proliferation and breast tumor growth in a strong RB-dependent fashion. HMGB1 also causes a RB-dependent G1 cell cycle arrest and induction of apoptosis. However, mutant HMGB1 with inactivating LXCXE motif (HMGB1-RXRXH) or missing LXCXE motif (HMGB1 delta LXCXE) becomes defective in these suppression functions. Thus, we hypothesize that HMGB1 interaction with RB mediates the suppression function in breast cancer, the disruption of the interaction results in defective activities of HMGB1. To test this hypothesis, we will carry out two specific aims. In Specific Aim 1, we will in detail examine the protein-protein interaction between HMGB1 and RB; determine the binding sites on each of HMGB1 and RB proteins; and evaluate the role of LXCXE motif in the interaction. We will determine three-dimensional structure of the HMGB1 protein for RB binding to identify the molecular features of the interaction. In Specific Aim 2, we will extend our preliminary studies to examine the effects of wild-type HMGB1 and RB-binding defective HMGB1 mutants on cell growth, cell cycle progression, apoptosis induction and telomerase activity in human breast cancer. These studies will provide a solid foundation for our long-term goal to discover HMGB1 as a novel tumor suppressor in breast cancer and to design new therapeutics for the prevention and treatment of breast cancer by modulating HMGB1-RB interaction and HMGBl-related gene therapy.